1. Field of the Invention
This invention relates to an agent for treating acquired immunodeficiency syndrome (AIDS), preventing AIDS virus infection and preventing the onset of AIDS after such infection.
2. Description of the Prior Art
AIDS is now a focus of worldwide attention as a disease induced by human immunodeficiency virus (HIV, or AIDS virus) and characterized by bad prognosis.
When infected with HIV, helper T cells, among others, are destroyed. Insufficiency of immunological competence thus results and this leads to such clinical features as serious opportunistic infection, carinii pneumonia, Kaposi sarcoma and AIDS-related encephalopathy. The disease is highly fatal.
HIV infection-associated states or illnesses include a symptomatic carrier state, progressive or persistent generalized lymphoadenophathy (PGL), lymphoadenophathy syndrome (LAD), AIDS-related complex (ARC) and AIDS. No effective therapy for AIDS has been established as yet. The only agent known to be effective is 3'-azidothymidine that has been shown to have obvious life-prolonging effect in patients with AIDS who are suffering from carinii pneumonia.
AZT is an HIV reverse transcriptase inhibitor and this effect brings about improvements in clinical symptoms and neurologic symptoms and temporary restoration of certain immune functions H. Mitsuya et al.; Nature, 325, 773 (1987)!. Therefore, AZT is highly toxic to bone marrow and about 50% of patients treated with AZT require blood transfusion.
No virucidal agent capable of specifically killing HIV without causing any serious adverse reactions in humans has not been discovered as yet.
Reverse transcriptase inhibitors, such as AZT, cannot be considered to be potent therapeutic agents since they produce adverse effects and are effective only in the postponing of death.
Accordingly, it is an object of the invention to provide an agent for treating AIDS and preventing HIV infection or the onset of AIDS after HIV infection.
The present inventors made investigations into the interactions between AIDS virus and T cells or, in other words, the sites of infection.
AIDS virus has an envelope glycoprotein (gp-120). Investigations have shown that gp-120 has a T cell receptor (CD-4)-binding site within the amino acid sequence from the 397th (from the N terminus of gp-120) to the 439th amino acid thereof A. L. Lawrence et al.: Cell, 50, 975 (1987)!.
According to more recent findings, the 24 amino acids (called epitope .beta.) from the 308th (from the N terminus) to the 332th amino acid of gp-120 plays an important role in HIV infection T. J. Palker: Proc. Natl. Acad. Sci. U.S.A., 85, 1932 (1988); S. Matsushita: J. Virol., 62 (6), 2107 (1988)!. That is to say, researchers, inclusive of the present inventors, have revealed that monoclonal antibodies to epitope .beta. of gp-120 can inhibit HIV infection.
Accordingly, the present inventors searched for proteins having an amino acid sequence homologous to epitope .beta. using the National Biomedical Research Foundation data base. As a result, 90 proteins were listed, 11 of which were proteases or protease inhibitors.
Among them, inter-.alpha.-trypsin inhibitors (ITIs), not only human ITI but also ITIs derived from various animals, showed that highest degree of homology to epitope .beta..
The arginine residue (Arg) in the homologous region was the protease activity inhibiting site of ITIs.
While epitope .beta. is the variable region of gp-120, the amino acid sequence comprising several amino acids with Arg as the central figure was preserved in various HIV-1 strains. Therefore, the present inventors synthesized a peptide having 33 amino acids in its amino acid sequence, including the 54 amino acids of epitope .beta. and evaluated it for trypsin activity inhibition by it. As a result, it was found that said peptide could inhibit trypsin activity by 30% when BOC-Phe-Ser-Arg-MCA was used as a synthetic substrate.
It was thus suggested that epitope .beta. might have protease inhibitor activity or be a very good substrate for protease.
Human ITI is a glycoprotein occurring in human serum. It is a single-chain glycoprotein having an apparent molecular weight of about 180,000 as determined by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and a carbohydrate content of about 8%. It is stable to acids and heat.
On the other hand, human urine trypsin inhibitor (hereinafter referred to as "UTI") purified from human urine is a single-chain glycoprotein having an apparent molecular weight of about 45,000 as determined by SDS-PAGE. It is stable to heat and acids.
Since both have the identical amino acid sequence on the N-terminal side, the ITI is presumably a precursor to UTI ("Proteinase Inhibitors", edited by A. J. Barrett et al., pages 389-398, Elsevier, 1986). UTI has two kunitz type domains closely resembling each other within its molecule. Nothing is known at all about what action, if any, said ITI and/or UTI can exert upon AIDS virus, however.
In Japan, human UTI, also called urinastatin by generic name, is currently in wide clinical use as a drug for the treatment of acute pancreatitis and acute ventricular failure (hemorrhagic shock, septic shock, traumatic shock, burn shock).
Since it is a glycoprotein derived from human urine, UTI is a very highly safe drug, scarcely presenting antigenicity, toxicity and other problems to humans.
For these reasons, the present inventors performed an in vitro infection neutralization test to see whether UTI might be actually effective against HIV infection.
The inhibition of HIV infection was assessed by the syncytium formation inhibition method, namely using, as an index, the syncytium formation inhibition in a mixed cell culture system containing LAV-1-infected CCRF-CEM cells and AIDS infection-free MOLT-4 clone 8 cells.
In this way, UTI was found to inhibit, in a serum-free culture system, syncytium formation strongly at concentrations not lower than 300 .mu.M, moderately at a concentration of 100 .mu.M, and weakly at 30 .mu.M. Soybean trypsin inhibitor (SBTI) was also tested by the above method since epitope .beta. shows homology, though weak, to the active site of SBTI. SBTI inhibited syncytium formation in a concentration-dependent manner at concentrations of 300 .mu.M to 3 mM, although its inhibitory activity was weaker than that of UTI.
On the contrary, aprotinin, which is a bovine lung-derived trypsin inhibitor, did not inhibit syncytium formation.
SBTI and aprotinin, which are heterologous proteins other than human-derived proteins, offer the antigenicity problem when they are repeatedly administered to humans. On the other hand, UTI can be considered to be an agent having very low toxicity. In fact, in acute toxicity testing in mice, rats and dogs, an intravenous dose of 150.times.10.sup.4 IU/kg body weight (i.e. about 600 mg/kg body weight) gave no deaths.
In subacute toxicity testing, where a maximum daily dose of 60.times.10.sup.4 IU/kg body weight (i.e. about 240 mg/kg body weight) was intravenously administered to rats and a maximum daily dose of 30.times.10.sup.4 IU/kg body weight (i.e. about 120 mg/kg body weight) to dogs for consecutive 4 weeks, no serious adverse reactions were noted.